U.S. patent application number 14/825613 was filed with the patent office on 2016-02-25 for anti-cancer extract and compounds.
The applicant listed for this patent is National Yang-Ming University. Invention is credited to Chi-Ying HUANG.
Application Number | 20160051608 14/825613 |
Document ID | / |
Family ID | 45891980 |
Filed Date | 2016-02-25 |
United States Patent
Application |
20160051608 |
Kind Code |
A1 |
HUANG; Chi-Ying |
February 25, 2016 |
ANTI-CANCER EXTRACT AND COMPOUNDS
Abstract
The present invention relates to a new approach for treating a
cancer or fibrosis, such as hepatocellular carcinoma, or liver
fibrosis using an extract from a plant of Graptopetalum sp.,
Rhodiola sp., or Echeveria sp., and prepared by extracting the
plant with dimethyl sulfoxide (DMSO), its fraction or the compound
isolated from the extract.
Inventors: |
HUANG; Chi-Ying; (Taipei,
TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National Yang-Ming University |
Taipei |
|
TW |
|
|
Family ID: |
45891980 |
Appl. No.: |
14/825613 |
Filed: |
August 13, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14189685 |
Feb 25, 2014 |
9132158 |
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14825613 |
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13249904 |
Sep 30, 2011 |
8686030 |
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14189685 |
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Current U.S.
Class: |
424/725 |
Current CPC
Class: |
A61K 36/41 20130101;
A61P 43/00 20180101; A61P 35/00 20180101; A61K 2236/30 20130101;
A61P 1/16 20180101 |
International
Class: |
A61K 36/41 20060101
A61K036/41 |
Claims
1. An extract with anti-cancer activity, which is extracted with
dimethyl sulfoxide (DMSO) from a plant of Graptopetalum sp.,
Rhodiola sp. or Echeveria sp.
2. The extract of claim 1, wherein the plant is Graptopetalum
paraguayense or Rhodiola rosea.
3. The extract of claim 1, which has a therapeutic effect on liver
cancer.
4. The extract of claim 1, which has a therapeutic effect on
hepatocellular carcinoma (HCC).
5. A composition comprising a therapeutically effective amount of
the extract of claim 1.
6. A method for preventing or treating a cancer comprising
administering to a subject in need thereof a therapeutically
effective amount of the extract of claim 1.
7. The method of claim 6, wherein the cancer is liver cancer.
8. The method of claim 6, wherein the liver cancer is
hepatocellular carcinoma (HCC).
Description
CROSS-REFERENCES
[0001] This application is a divisional application of U.S. patent
application Ser. No. 14/189,685 by Chi-Ying Huang, entitled
"Anti-Cancer Extract and Compounds," and filed on Feb. 25, 2014,
which in turn is a divisional application of U.S. patent
application Ser. No. 13/249,904 by Chi-Ying Huang, entitled
"Anti-Cancer Extract and Compounds," and filed on Sep. 20, 2011,
now U.S. Pat. No. 8,686,030; the contents of both of these
applications are incorporated herein in their entirety by this
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a new extract and new
compounds having anti-cancer activities from a plant.
BACKGROUND OF THE INVENTION
[0003] Graptopetalum paraguayense (GP) is a Chinese traditional
herb and possesses several health benefits. According to its
archaic Chinese prescription, GP is considered to have potentially
beneficial effects by alleviating hepatic disorders, lowering blood
pressure, whitening skin, relieving pain and infections, inhibiting
inflammation, and improving brain function.
[0004] It was shown in the studies that the leaf extracts of GP
could inhibit tyrosinase and angiotensin-converting enzyme
activities and scavenge free radicals in vitro (Chen, S-J et al.,
Studies on the inhibitory effect of Graptopetalum paraguayense E.
Walther extracts on the angiotensin converting enzyme. Food
Chemistry 100:1032-1036, 2007; Chung, Y-C et al., Studies on the
antioxidative activity of Graptopetalum paraguayense E. Walther.
Food Chemistry 91:419-424, 2005; and Huang, K-F et al., Studies on
the inhibitory effect of Graptopetalum paraguayense E. Walther
extracts on mushroom tyrosinase. Food Chemistry 89:583-587, 2005.)
It was found that the water and 50% ethanolic and 95% ethanolic
stem extracts of GP has antioxidant activity, which were assayed
for inhibitory effects on the proliferation of a human HCC cell
line (HepG2) (Chen, S J et al., In vitro antioxidant and
antiproliferative activity of the stem extracts from Graptopetalum
paraguayense. Am J Chin Med 36:369-383, 2008). An in vivo research
study demonstrated that the leaf extracts of GP inhibited microglia
activation, oxidative stress, and iNOS expression to reduce
ischemic brain injury (Kao, T K et al., Graptopetalum paraguayense
E. Walther leaf extracts protect against brain injury in ischemic
rats. Am J Chin Med 38:495-516, 2010.).
[0005] It was disclosed in U.S. Pat. No. 7,364,758 filed in 2004 by
Hsu and granted in 2008 that the ethanolic extract from
Graptopetalum had anti-liver fibrosis and anti-inflammatory effects
in vivo and in vitro. Then, its continuation-in-part application,
U.S. Pat. No. 7,588,776, was filed in 2008 and granted in 2009
indicating that the water-soluble fraction of Graptopetalum was
effective in treating a liver disease or condition, such as
inflammation, steatosis, and fibrosis.
SUMMARY OF THE INVENTION
[0006] The present invention relates to a new extract and its
fraction, and new compounds, which are isolated from a plant,
particularly Graptopetalum sp. The invention also provides a new
approach for treating a cancer, particularly Hepatocellular
carcinoma (HCC), using the new extract or the new compounds.
[0007] In one aspect, the invention provides an extract with
anti-cancer activity, which is extracted with Dimethyl sulfoxide
(DMSO) from a plant selected from the group consisting of
Graptopetalum sp., Rhodiola sp. and Echeveria sp. It is
unexpectedly found in the present invention that the DMSO extract
has anti-cancer activity.
[0008] In one embodiment of the invention, the plant is
Graptopetalum paraguayense or Rhodiola rosea. In one example of the
invention, the extract is obtained by extracting the plant with 30%
DMSO.
[0009] In another aspect, the invention provides a fraction
containing rich anti-cancer components from a plant selected from
the group consisting of Graptopetalum sp., Rhodiola sp. and
Echeveria sp, particularly Graptopetalum paraguayense or Rhodiola
rosea, which is prepared by extracting the plant with Dimethyl
sulfoxide (DMSO), and then isolating by chromatography to obtain a
fraction called as HH-F3, which has effects in causing cytotoxicity
and down-regulating AURKA, AURKB, and F1110540 expression in cancer
cells.
[0010] In one embodiment of the invention, a Sephadex LH-20 column
was used as a chromatography column. According to the invention,
the fraction according to the invention has high cytotoxicity
effects and is effective in down-regulating AURKA, AURKB, and
FLJ10540 expression in Huh7 and HepG2 cells.
[0011] In the mechanistic study on the fraction HH-F3, it was found
that the fraction HH-F3 induced HCC to undergo apoptosis. In other
words, it was indicated that the fraction HH-F3 has a therapeutic
effect on cancer cells, particularly HCC.
[0012] In a further aspect, the invention provides a compound,
having a structure of formula I below,
##STR00001##
wherein one of the Rs is H, or a prucyanidin (PC) unit; and the
other is OH or a prodelphindine (PD) unit; n is a number ranging
from 21 to 38; and PC unit:PD unit<1:20. The structure of
prucyanidin (PC) unit is
##STR00002##
and the structure of prodelphindine (PD) is
##STR00003##
[0013] According to the invention, the compound of formula I can be
isolated from a plant selected from the group consisting of
Graptopetalum sp., Rhodiola sp. or Echeveria sp. In one embodiment
of the invention, the compound was purified from the fraction of
Graptopetalum paraguayense or Rhodiola rosea. It was found that the
compound of formula I is rich in 3,4,5-trihydroxy benzylic
moieties, and has anti-cancer activities.
[0014] In yet another aspect, the invention provides a composition
or a pharmaceutical composition, comprising the extract, the
fraction or the compound of the invention, and a pharmaceutically
acceptable carrier. Furthermore, the pharmaceutical composition has
anti-cancer activity, which is effective in the prevention or
treatment of a cancer, such as liver cancer particularly HCC.
[0015] In further yet aspect, the invention provides the use of the
extract, the fraction or the compound of formula I of the invention
in manufacture of a medicament for treating a cancer, particularly
HCC.
[0016] In still another aspect, the invention provides a method for
preventing or treating a cancer, comprising administering to a
subject in need thereof a therapeutically effective amount of the
extract, the fraction or the compound of the invention. In one
example of the invention, the cancer is HCC.
[0017] The foregoing summary, as well as the following detailed
description of the will be better understood when read in
conjunction with the appended drawings. For the purpose of
illustrating the invention, there are shown in the drawings the
embodiments which are presently preferred. It should be understood,
however, that the invention is not limited to the embodiments shown
in the drawings.
[0018] In the drawings:
[0019] FIG. 1 provides a HPLC fingerprint of the fraction HH-F3
according to the invention, wherein the chromatogram and the
elution gradient curve were marked as Line X and Line Y,
respectively.
[0020] FIG. 2 shows the effects of the fraction HH-F3 according to
the invention in causing an increase of cytotoxicity in Huh7 cells
as compared with other extracts prepared from different solvents,
wherein Huh7 cells were treated with GP extracts prepared from
water, acetone, methanol, 100% ethanol, 70% ethanol, 50% ethanol,
100% DMSO, and 30% DMSO at the concentrations of 100, 150, 250,
500, 750, 1000, and 1500 .mu.g/mL for 72 hours, after which the
cells were subjected to MTT assays. Of these extract preparations,
the 30% DMSO GP extracts exhibited the most significant inhibition
of cell viability in Huh7 cells at 72 hours.
[0021] FIG. 2 also shows the effects of the fraction HH-F3
according to the invention in causing an increase of cytotoxicity
in Mahlavu cells as compared with other extracts prepared from
different solvents, wherein Mahlavu cells were treated with GP
extracts prepared from water, acetone, methanol, 100% ethanol, 70%
ethanol, 50% ethanol, 100% DMSO, and 30% DMSO at the concentrations
of 100, 150, 250, 500, 750, 1000, and 1500 .mu.g/mL for 72 hours,
after which the cells were subjected to MTT assays. Of these
extract preparations, the 30% DMSO GP extracts exhibited the most
significant inhibition of cell viability in Mahlavu cells at 72
hours.
[0022] FIG. 3 shows the effects of the fraction HH-F3 according to
the invention in decreasing degradation of several mitotic
regulators during interphase and M phase in HSC-T6 cells wherein
HSC-T6 cells were treated with the 30% DMSO GP extracts. The cell
lysates were subjected to immunoblot analysis with anti-FLJ10540
and anti-AURKB antibodies.
[0023] FIG. 3 also shows the effects of the fraction HH-F3
according to the invention in decreasing degradation of several
mitotic regulators during interphase and M phase in HepG2 and Huh7
cells that were treated with various concentrations of the 30% DMSO
GP extracts; the cell lysates were subjected to immunoblot analysis
with anti-FLJ10540, anti-AURKA, and anti-AURKB antibodies.
[0024] FIG. 3 further shows the effects of the fraction HH-F3
according to the invention in decreasing degradation of several
mitotic regulators during interphase and M phase in HepG2 and Huh7
cells, wherein HepG2 and Huh7 cells were treated with 75 ng/mL
nocodazole (NOC) for 16-18 hours. After pretreatment with the
synchronizing agent, the cells were then treated with 750 .mu.g/mL
30% DMSO GP extracts or vehicle control (30% DMSO) for another 3
hours; Western blots were performed using anti-F1110540,
anti-AURKA, and anti-AURKB antibodies; it is important to note the
following: (1) AURKA, AURKB, and FLJ10540 were highly expressed in
mitotic cells compared with interphase cells, and (2) the protein
expression levels of AURKA, AURKB, and FLJ10540 in interphase and
metaphase were both suppressed after treatment with the 30% DMSO GP
extracts.
[0025] FIG. 4 shows the effects of the fraction HH-F3 according to
the invention in suppressing AURKA protein expression in the HCC
cell lines (Huh7 cells) wherein Huh7 cells were treated with GP
extracts that were prepared from water, acetone, methanol, 100%
ethanol, 70% ethanol, 50% ethanol, 100% DMSO, 30% DMSO at a
concentration of 500 .mu.g/mL for 48 hours; and AURKA expression
levels were inhibited after treatment with the 30% DMSO GP
extracts.
[0026] FIG. 4 also shows the effects of the fraction HH-F3
according to the invention in suppressing AURKA protein expression
in the HCC cell lines (HepG2 and Huh7 cells) wherein AURKA, AURKB,
and F1110540 expression levels were not suppressed by the water and
BuOH fractions in the HepG2 and Huh7 cells.
[0027] FIG. 5 shows the time- and dose-dependent response in
causing cytotoxicity of the fraction HH-F3 according to the
invention in Huh7 cells wherein Huh7 (FIG. 5A) cells were treated
with the 30% DMSO GP extracts at concentrations of 0, 250, 500,
750, and 1000 .mu.g/mL for 24, 48, and 72 hours, followed by MTT
assays. The IC.sub.50 values for growth inhibition caused by the
30% DMSO GP extracts in Huh7 cells were approximately 500 .mu.g/mL
at 48 hours.
[0028] FIG. 5 also shows the time- and dose-dependent response in
causing cytotoxicity of the fraction HH-F3 according to the
invention in Mahlavu cells wherein Mahlavu cells were treated with
the 30% DMSO GP extracts at concentrations of 0, 250, 500, 750, and
1000 .mu.g/mL for 24, 48, and 72 hours, followed by MTT assays. The
IC.sub.50 values for growth inhibition caused by the 30% DMSO GP
extracts in Mahlavu cells were approximately 250 .mu.g/mL at 48
hours.
[0029] FIG. 6 shows the effects of the different purification
fractions of GP on AURKA protein expression in the HCC cell lines,
illustrating the purification scheme of the GP extract and fraction
HH-F3 according to the invention.
[0030] FIG. 6 further shows that HepG2 cells were treated with the
30% DMSO GP extracts, HH-F1, HH-F2, HH-F3, and HH-F4 for 3 hours.
AURKA and AURKB expression levels were not suppressed by treatment
with the HH-F1, HH-F2 and HH-F4 fractions in HepG2 cells.
[0031] FIG. 6 further shows that the expression of AURKA was
inhibited after treatment with the 30% DMSO GP extracts and the
HH-F3 fraction.
[0032] FIG. 6 also shows that the expression of AURKA was inhibited
after treatment with the HH-F3a fraction.
[0033] FIG. 7 shows the effects of the fraction HH-F3 according to
the invention in inhibition of Huh7 cells, wherein Huh7 cells were
treated with the HH-F3 fraction at concentrations of 5, 25, 50, and
75 .mu.g/mL for 24, 48, and 72 hours, followed by MTT assays. The
IC.sub.50 value for the inhibition of cell viability caused by
treatment with the HH-F3 fraction in Huh7 cells was approximately
50 .mu.g/mL after treatment for 72 hours.
[0034] FIG. 7 further shows the effects of the fraction HH-F3
according to the invention in inhibition of Mahlavu cells, wherein
Mahlavu cells were treated with the HH-F3 fraction at
concentrations of 5, 25, 50, and 75 .mu.g/mL for 24, 48, and 72
hours, followed by MTT assays. The IC.sub.50 value for the
inhibition of cell viability caused by treatment with the HH-F3
fraction in Mahlavu cells was approximately 37.5 .mu.g/mL after
treatment for 72 hours.
[0035] FIG. 7 further shows the effects of the fraction HH-F3
according to the invention in inhibition of PLC5 cells, wherein
PLC5 cells were treated with the HH-F3 fraction at concentrations
of 5, 25, 50, and 75 .mu.g/mL for 24, 48, and 72 hours, followed by
MTT assays. The IC.sub.50 value for the inhibition of cell
viability caused by treatment with the HH-F3 fraction in PLC5 cells
was approximately 75 .mu.g/mL after treatment for 72 hours.
[0036] FIG. 7 further shows the effects of the fraction HH-F3
according to the invention in inhibition of HSC-T6 cells, wherein
HSC-T6 cells were treated with the HH-F3 fraction at concentrations
of 5, 25, 50, and 75 .mu.g/mL for 24, 48, and 72 hours, followed by
MTT assays. The IC.sub.50 value for the inhibition of cell
viability caused by treatment with the HH-F3 fraction in HSC-T6
cells was approximately 20 .mu.g/mL after treatment for 72
hours.
[0037] FIG. 7 also shows the effects of the fraction HH-F3
according to the invention in inhibition of Huh7 cells, wherein
Huh7 cells were treated with the HH-F3 fraction at concentrations
of 5, 25, 50, and 75 .mu.g/mL for 24, 48, and 72 hours, followed by
trypan blue assays.
[0038] FIG. 7 also further shows the effects of the fraction HH-F3
according to the invention in inhibition of Mahlavu cells, wherein
Mahlavu cells were treated with the HH-F3 fraction at
concentrations of 5, 25, 50, and 75 .mu.g/mL for 24, 48, and 72
hours, followed by trypan blue assays.
[0039] FIG. 7 also further shows the effects of the fraction HH-F3
according to the invention in inhibition of PLC5 cells, wherein
PLC5 cells were treated with the HH-F3 fraction at concentrations
of 5, 25, 50, and 75 .mu.g/mL for 24, 48, and 72 hours, followed by
trypan blue assays.
[0040] FIG. 8 shows that the HH-F3 fraction down-regulates AURKA
and F1110540 in HCC cell lines and activated hepatic stellate
cells. In FIG. 8, Huh7, Mahlavu, and PLC5 cells were treated with
the HH-F3 fraction at concentrations of 25, 50, or 75 .mu.g/mL for
3 hours. Expression of both AURKA and FLJ10540 was down-regulated
in a concentration-dependent manner, as examined by immunoblot
analysis with anti-FLJ10540 and anti-AURKA antibodies.
[0041] FIG. 8 further shows that the HH-F3 fraction down-regulates
AURKA and F1110540 in HCC cell lines and activated hepatic stellate
cells. In FIG. 8, HSC-T6 cells were treated with the HH-F3 fraction
at concentrations of 5, 15, and 50 .mu.g/mL for 3 hours; FLJ10540
expression was down-regulated in a concentration-dependent
manner.
[0042] FIG. 9 shows the effect of the fraction HH-F3 in causing
apoptosis in HHC cell lines; Huh7 cells were treated with 5, 25,
and 50 .mu.g/mL HH-F3 for 24 and 48 hours. After treatment with the
HH-F3 fraction for 24 and 48 hours (FIG. 9), the cell lysates were
subjected to immunoblot analysis for anti-cleaved caspase-3 and
cleaved PARP. FIG. 9 shows the results for Huh7 cells.
[0043] FIG. 9 further shows the effect of the fraction HH-F3 in
causing apoptosis in HHC cell lines; Mahlavu cells were treated
with 5, 25, and 50 .mu.g/mL HH-F3 for 24 and 48 hours. After
treatment with the HH-F3 fraction for 24 and 48 hours, the cell
lysates were subjected to immunoblot analysis for anti-cleaved
caspase-3 and cleaved PARP. FIG. 9 shows the results for Mahlavu
cells.
[0044] FIG. 10 shows the effect of the fraction HH-F3 in decreasing
mitochondrial membrane potential and increasing ROS generation in
the HCC cell lines; mitochondria membrane potential (.DELTA..PSI.)
of Huh7 cells was analyzed using the JC-1 mitochondrial membrane
potential assay, and the AT of the cells decreased after treatment
with the fraction HH-F3 at the concentrations of 5, 10, 15, 25, and
50 .mu.g/mL for 48 hours (n=2); RFU=AT.
[0045] FIG. 10 further shows the effect of the fraction HH-F3 in
decreasing mitochondrial membrane potential and increasing ROS
generation in the HCC cell lines; mitochondria membrane potential
(.DELTA..PSI.) of Mahlavu cells was analyzed using the JC-1
mitochondrial membrane potential assay, and the AT of the cells
decreased after treatment with the fraction HH-F3 at the
concentrations of 5, 10, 15, 25, and 50 .mu.g/mL for 48 hours
(n=2); RFU=AT.
[0046] FIG. 10 also shows the effect of the fraction HH-F3 in
decreasing mitochondrial membrane potential and increasing ROS
generation in the HCC cell lines for Huh7 cells; intracellular
superoxide (O.sub.2.sup.-) levels, as measured by hydroethidine
(HE) staining, were decreased significantly 48 hours after
treatment with the fraction HH-F3 at the concentrations of 5, 10,
15, 25, and 50 .mu.g/mL as compared with the control HCC cells
(Huh7 cells treated with DMSO).
[0047] FIG. 10 also further shows the effect of the fraction HH-F3
in decreasing mitochondrial membrane potential and increasing ROS
generation in the HCC cell lines for Mahlavu cells; intracellular
superoxide (O.sub.2.sup.-) levels, as measured by hydroethidine
(HE) staining, were decreased significantly 48 hours after
treatment with the fraction HH-F3 at the concentrations of 5, 10,
15, 25, and 50 .mu.g/mL as compared with the control HCC cells
(Mahlavu cells treated with DMSO).
[0048] FIG. 10 also further shows that intracellular peroxide
levels, as measured by DCFH, were increased 48 hours after
treatment with the fraction HH-F3 at the concentrations of 5, 10,
15, 25, and 50 .mu.g/mL for Huh7 cells as compared with control HCC
cells (Huh7 treated with DMSO), and it was found that the
production of intracellular peroxide and superoxide increased in a
dose-dependent manner in Huh7 cells after treatment with the
fraction HH-F3.
[0049] FIG. 10 also further shows that intracellular peroxide
levels, as measured by DCFH, were increased 48 hours after
treatment with the fraction HH-F3 at the concentrations of 5, 10,
15, 25, and 50 .mu.g/mL for Mahlavu cells as compared with control
HCC cells (Huh7 treated with DMSO), and it was found that the
production of intracellular peroxide and superoxide increased in a
dose-dependent manner in Mahlavu cells after treatment with the
fraction HH-F3.
[0050] FIG. 11 shows the effect of the fraction HH-F3 in inhibiting
AKT-Ser.sup.473 phosphorylation and activating PTEN protein
expression in Huh7 cells that were treated with the fraction HH-F3
at the concentrations of 25, 50, or 75 .mu.g/mL for 48 hours, and
the expression of AURKA, FLJ10540, AKT-Ser.sup.473 was
down-regulated, whereas PTEN was up-regulated in a
concentration-dependent manner, as examined by immunoblot analysis
with anti-F1 LJ0540, anti-AURKA, anti-AKT-Ser.sup.473, and
anti-PTEN antibodies.
[0051] FIG. 12 shows the effects of the GP extract and the fraction
HH-F3 according to the invention in decreasing the hydroxyproline
content in cirrhotic liver and tumor burdens; wherein the animals
were divided into four groups; they were provided with tap water
only (normal group) or with DEN solution (the other group), as
described in the Materials and Methods. FIG. 12 shows the decreased
bile flow in cirrhotic animals, which was recorded to measure liver
function (*P<0.05; ANOVA).
[0052] FIG. 12 further shows the effects of the GP extract and the
fraction HH-F3 according to the invention in decreasing the
hydroxyproline content in cirrhotic liver and tumor burdens;
wherein the animals were divided into four groups; they were
provided with tap water only (normal group) or with DEN solution
(the other group), as described in the Materials and Methods. FIG.
12 shows the enlarged spleen size in cirrhotic animals wherein the
spleen weights and body weights (BWs) were measured and expressed
as spleen weight/BW; the ratios of spleen weight/BW of the DEN
group was significantly higher than those of the normal group,
which indicates that the splenomegaly was due to cirrhosis-related
portal hypertension (P<0.05, ANOVA), whereas only the ratio of
spleen weight/BW of the high-dose group was significantly lower
than those of the DEN group (P<0.05, ANOVA).
[0053] FIG. 12 also shows the effects of the GP extract and the
fraction HH-F3 according to the invention in decreasing the
hydroxyproline content in cirrhotic liver and tumor burdens;
wherein the animals were divided into four groups; they were
provided with tap water only (normal group) or with DEN solution
(the other group), as described in the Materials and Methods. FIG.
12 shows the increased collagen content in cirrhotic livers,
wherein liver cirrhosis was determined by measuring the levels of
liver hydroxyproline content and significant decreases were
observed when comparing the high-dose group, the HH-F3 group and
the control group.
[0054] FIG. 12 also further shows the effects of the GP extract and
the fraction HH-F3 according to the invention in decreasing the
hydroxyproline content in cirrhotic liver and tumor burdens;
wherein the animals were divided into four groups; they were
provided with tap water only (normal group) or with DEN solution
(the other group), as described in the Materials and Methods. FIG.
12 shows the expression of .alpha.-SMA induced by DEN. The
formalin/paraffin sections of the liver samples from each group
during the course of DEN feeding stained with an antibody against
.alpha.-SMA, the percentages of .alpha.-SMA (+) area were
determined by a Digital Camera System using the 10 fields with the
densest staining (*P<0.05; **P<0.005, ANOVA).
[0055] FIG. 12 also further shows the effects of the GP extract and
the fraction HH-F3 according to the invention in decreasing the
hydroxyproline content in cirrhotic liver and tumor burdens;
wherein the animals were divided into four groups; they were
provided with tap water only (normal group) or with DEN solution
(the other group), as described in the Materials and Methods. FIG.
12 shows the oxidative stress induced by DEN. NBT (Nitrotetrazolium
blue chloride) is a dye that is reduced to an insoluble
blue-colored formazan derivative upon exposure to superoxide, and
the blue-colored deposit as a histological marker for the presence
of superoxide in tissue is detectable by light microscopy, the
density of NBT (+) foci was determined, as described in the
Materials and Methods, from the 10 fields with the densest
staining.
[0056] FIG. 12 still further shows the effects of the GP extract
and the fraction HH-F3 according to the invention in decreasing the
hydroxyproline content in cirrhotic liver and tumor burdens;
wherein the animals were divided into four groups; they were
provided with tap water only (normal group) or with DEN solution
(the other group), as described in the Materials and Methods. FIG.
12 shows the measurement of tumor burdens wherein the livers
obtained from the sacrificed animals were sliced into 5-mm
sections, the numbers and sizes of all visible tumor nodules with
diameters larger than 3 mm were counted and measured. Tumor burdens
are expressed as the sum of the volume of total tumor nodules
(**P<0.005, ***P<0.001 as compared to the DEN group).
[0057] FIG. 12 still further shows the effects of the GP extract
and the fraction HH-F3 according to the invention in decreasing the
hydroxyproline content in cirrhotic liver and tumor burdens;
wherein the animals were divided into four groups; they were
provided with tap water only (normal group) or with DEN solution
(the other group), as described in the Materials and Methods. FIG.
12 shows the gross picture of chemical-induced HCC and cirrhosis,
wherein the 9 weeks of oral administration of DEN in drinking water
on the rat livers resulted in multiple hepatic tumors in the
cirrhotic rat livers, and the development of granulation on the
surface and the uneven boundary with multiple hepatic tumors was
observed in these animals.
[0058] FIG. 13 shows the effect of the Rhodiola rosea extract in
inhibiting the cell viability of the HCC cell lines (PLC5, Huh7,
and Mahlavu) and down-regulating AURKA protein expression. FIG. 13
shows the effect of the extract on inhibiting the cell
viability.
[0059] FIG. 13 further shows the effect of the Rhodiola rosea
extract in inhibiting the cell viability of the HCC cell lines
(PLC5, Huh7, and Mahlavu) and down-regulating AURKA protein
expression. FIG. 13 shows the effect on the extract on AURKA
expression, with .beta.-actin as a control.
DETAILED DESCRIPTION
[0060] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by a
person skilled in the art to which this invention belongs.
[0061] As used herein, the singular forms "a", "an", and "the"
include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to "a sample" includes a
plurality of such samples and equivalents thereof known to those
skilled in the art.
[0062] The invention provides a new extract of a plant selected
from the group consisting of Graptopetalum sp., Rhodiola sp. or
Echeveria sp., prepared by extracting the plant with DMSO, referred
to as the GP extract. It is unexpectedly found in the present
invention that the GP extract has anti-cancer activity.
[0063] According to the invention, the extract may be prepared by
extracting the plant with dimethyl sulfoxide (DMSO) using commonly
used or standard methods in this art. In one example of the
invention, the leaves of the plant are grounded and lyophilized
into powder and vortexed with DMSO, preferably 30% DMSO. A further
extraction with methanol (MeOH) may be included before the
extraction with DMSO.
[0064] The term "Graptopetalum", as used herein, refers to any
plant in the genus of Graptopetalum, or part or parts thereof.
Combinations of more than one species of Graptopetalum, or parts
thereof, are also contemplated. The Graptopetalum is preferably
Graptopetalum paraguayense.
[0065] The term "Rhodiola ", as used herein, refers to any plant in
the genus of Rhodiola, or part or parts thereof. Combinations of
more than one species of Rhodiola, or parts thereof, are also
contemplated. The Rhodiola is preferably Rhodiola rosea.
[0066] The term "Echeveria", as used herein, refers to any plant in
the genus of Echeveria, or part or parts thereof. Combinations of
more than one species of Echeveria, or parts thereof, are also
contemplated. The Echeveria is preferably Echeveria peacockii.
[0067] In one preferred embodiment of the invention, the plant is
Graptopetalum paraguayense or Rhodiola rosea.
[0068] The term "extract" as used herein refers to a solution
obtained by soaking or mixing a substance to be extracted with a
solvent. In the present invention, the extract is a DMSO
extract.
[0069] The invention also provides a fraction containing rich
anti-cancer components from a plant selected from the group
consisting of Graptopetalum sp., Rhodiola sp. and Echeveria sp,
which is prepared by extracting the plant with Dimethyl sulfoxide
(DMSO), and then isolating by chromatography to obtain a fraction
called as HH-F3. In one example of the invention, the plant is
Graptopetalum paraguayense or Rhodiola rosea. The fraction is
obtained by extracting the plant with DMSO and isolating by
chromatography to obtain a fraction, referred to as HH-F3. In one
example of the invention, a Sephadex LH-20 column is used for
chromatography. It was found that the fraction has effects in
causing cytotoxicity and down-regulating AURKA, AURKB, and F1110540
expression in cancer cells, particularly the HCC cell lines such as
Huh7 and HepG2 cells. A mechanistic study of the fraction HH-F3 was
performed and it was indicated that the fraction HH-F3 induced HCC
to undergo apoptosis. Accordingly, the fraction HH-F3 is a
potential therapeutic agent for the prevention or treatment of a
cancer, particularly liver cancer, such as HCC.
[0070] According to one example of the invention, a sub-fraction
referred to as HH-F3a was obtained from the fraction HH-F3 via
dialysis. Then, the active compounds were isolated from the HH-F3a
fraction, which is different from the known proanthocyanidin
compounds. The compound as obtained is a proanthocyanidin rich in
3,4,5-trihydroxy benzylic moieties. The compound is of a structure
of formula I below,
##STR00004##
wherein one of the Rs is H, or a prucyanidin (PC) unit; and the
other is OH or a prodelphindine (PD) unit; n is a number ranging
from 21 to 38; and PC unit:PD unit<1:20. The structure of a
prucyanidin (PC) unit is
##STR00005##
and the structure of prodelphindine (PD) is
##STR00006##
[0071] Accordingly, the invention provides the compound of formula
I, which is proved to have anti-cancer activity. In one example to
the invention, the compound of formula I was obtained by an
extraction from the plant of Graptopetalum sp., Rhodiola sp. or
Echeveria sp. with DMSO to obtain a DMSO extract, a selection from
the DMSO extract using down-regulation regulation of AURKA via
western blot to obtain a sub-fraction referred to as "HH-F3a" via
dialysis, and a further purification.
[0072] The invention provides a pharmaceutical composition,
comprising a therapeutically effective amount of the extract, the
fraction or the compound of formula I of the invention, and a
pharmaceutically acceptable carrier.
[0073] The term "therapeutically effective amount" as used herein
refers to an amount of an agent sufficient to achieve the intended
purpose for treatment. For example, an effective amount of
Graptopetalum to treat HCC is an amount sufficient to kill HCC
cells. The therapeutically effective amount of a given agent will
vary with factors such as the nature of the agent, the route of
administration, the size and species of the animal to receive the
agent, and the purpose of the administration. The therapeutically
effective amount in each individual case may be determined
empirically by a skilled artisan according to the disclosure herein
and established methods in the art. The pharmaceutical composition
of the invention may be administered in any route that is
appropriate, including but not limited to parenteral or oral
administration. The pharmaceutical compositions for parenteral
administration include solutions, suspensions, emulsions, and solid
injectable compositions that are dissolved or suspended in a
solvent immediately before use. The injections may be prepared by
dissolving, suspending or emulsifying one or more of the active
ingredients in a diluent. Examples of said diluents are distilled
water for injection, physiological saline, vegetable oil, alcohol,
and a combination thereof. Further, the injections may contain
stabilizers, solubilizers, suspending agents, emulsifiers, soothing
agents, buffers, preservatives, etc. The injections are sterilized
in the final formulation step or prepared by sterile procedure. The
pharmaceutical composition of the invention may also be formulated
into a sterile solid preparation, for example, by freeze-drying,
and may be used after sterilized or dissolved in sterile injectable
water or other sterile diluent(s) immediately before use.
[0074] According to the invention, the composition may also be
administered through oral tablets, pills, capsules, dispersible
powders, granules, and the like. The oral compositions also include
gargles which are to be stuck to oral cavity and sublingual
tablets. The capsules include hard capsules and soft capsules. In
such solid compositions for oral use, one or more of the active
compound(s) may be admixed solely or with diluents, binders,
disintegrators, lubricants, stabilizers, solubilizers, and then
formulated into a preparation in a conventional manner. When
necessary, such preparations may be coated with a coating agent, or
they may be coated with two or more coating layers. On the other
hand, the liquid compositions for oral administration include
pharmaceutically acceptable aqueous solutions, suspensions,
emulsions, syrups, elixirs, and the like. In such compositions, one
or more of the active compound(s) may be dissolved, suspended or
emulsified in a commonly used diluent (such as purified water,
ethanol or a mixture thereof, etc.). Besides such diluents, said
compositions may also contain wetting agents, suspending agents,
emulsifiers, sweetening agents, flavoring agents, perfumes,
preservatives and buffers and the like.
[0075] It was confirmed in the examples that the extract, the
fraction or the compound of the invention, or the pharmaceutical
composition thereof caused an apoptosis in HCC cells, suggesting
that they have anti-cancer activity, which may be used for the
prevention or treatment of a cancer, particularly HCC. On the other
hand, it is suggested that they are effective in treatment of
fibrosis, particularly liver fibrosis.
[0076] Accordingly, the invention provides the use of the extract,
the fraction or the compound of formula I of the invention in
manufacture of a medicament for treating a cancer, particularly,
HCC. On the other hand, the invention provides a method for
preventing or treating a cancer or fibrosis comprising
administering to a subject in need thereof a therapeutically
effective amount of the extract, the fraction or the compound of
the invention, particularly HCC and liver fibrosis.
[0077] The following examples are offered to illustrate this
invention and are not to be construed in any way as limiting the
scope of the present invention.
EXAMPLE
Example 1
Extraction and Purification from GP or Rhodiola rosea
[0078] The leaves of Graptopetalum paraguayense (referred to as GP)
were ground and lyophilized into powder at -20.degree. C. and
stored in a moisture buster at 25.degree. C. before extraction.
First, 1.5 g GP powder was vortexed with 10 mL 100% methanol (MeOH)
for 5 minutes and then centrifuged at 1500 g for 5 minutes. After
removal of the supernatant, 10 mL H.sub.2O, 100% acetone, 100%
methanol, 100% ethanol, 70% ethanol, 50% ethanol, 100% DMSO and 30%
DMSO was added to each pellet to resuspend them for each extract.
The suspension was mixed by vortexing for 5 minutes, centrifuged
twice at 1500 g for 5 minutes, centrifuged again at 9300 g for 5
minutes, and filtered using a 0.45 .mu.m filter by laminar flow at
room temperature. The 30% DMSO supernatant was either fractionated
into four fractions (FI-F4) by a Sephadex LH-20 20 column or stored
at -20.degree. C. as a 150 mg/mL stock solution (referred to as 30%
DMSO GP extracts). The GP extract or the fraction HH-F3 was also
subjected to dialysis against water by a dialysis membrane (MWCO
12-14,000) (Spectrum Laboratories, Rancho Dominguez, Calif.) to
obtain active compounds. Using the analysis of AURKA, AURKB, and
FLJ10540 protein levels via Western blot, active molecules were
analyzed, which we refer to as the fraction HH-F3. In addition, the
fraction HH-F3 was further analyzed by HPLC and .sup.1H- and
.sup.13C-NMR spectra to identify the structure of the active
molecules.
[0079] Similarly, the plants of Rhodiola rosea (referred to as RS)
were lyophilized into powder and stored in moisture buster at
25.degree. C. before extraction. One and a half grams of RS powder
was dissolved in 10 mL H.sub.2O and then centrifuged at 1500 g for
5 minutes, followed by filtering using a 0.45 .mu.m filter by
laminar flow at room temperature. The samples were stored at
-20.degree. C. as 150 mg/mL stock solutions.
[0080] A chemical investigation on the fraction HH-F3 of the
invention resulted in the identification of its major components as
polyphenolic compounds, according to broadened aromatic signals in
the .sup.1H and .sup.13C NMR spectra. The major compounds in the
HH-F3 fraction were identified to be tannins because of their
characteristic pink color with partial silver metal-like like
luster after lyophilization. The total tannin content of the HH-F3
fraction was approximately 68%, as determined by a colorimetric
assay for condensed tannin quantification, which used catechin as
the standard when monitored at OD.sub.500. The HPLC fingerprint of
the HH-F3 fraction (FIG. 1) revealed that two groups of compounds
(Groups A and B) with distinct molecular weight ranges existed in
the HH-F3 fraction, and one major and one minor component were
detected in Group B. A proanthocyanidin-rich high molecular weight
fraction, HH-F3a (with a yield of 71.9% compared to the amount of
the starting material HH-F3), was prepared from the HH-F3 fraction
using dialysis. Briefly, HH-F3 (112.1 mg) was dialyzed by a
dialysis membrane (MWCO 12-14,000) against water to give an inner
membrane fraction (HH-F3A, 80.6 mg) and an outer-membrane fraction
(7.4 mg). This fraction contained the active compounds, as
determined by measuring the disappearance of AURKA by Western blot
(FIG. 6). The main skeleton for the proanthocyanidin fraction of
HH-F3a was determined to be a proanthocyanidin polymer (see below),
and its physiochemical properties, including the mean molecular
weight (mMW), mean degree of polymerization (mDP), PC:PD ratio and
stereochemistry (cis:trans), are listed in Table 1. mMW and mDP
were determined by analyzing the ratio of the degraded terminal and
elongating monomers. In addition, to simplify the preparation
protocols, another method via directly dialysis of GP extracts
against water (Method II) was performed, and the physiochemical
data of the compounds prepared by Method II are also listed in
Table 1. The listed physiochemical properties in Table 1 suggest
that the fraction prepared by Method II was identical to that of
Method I (the method used to prepare HH-F3a).
##STR00007##
TABLE-US-00001 TABLE 1 Physicochemical Properties for the
Proanthocyanidin Polymer of Formula 1 According to the Invention
mMW Preparation PC:PD Cis:trans 3-O-galloyl mDP (kD) Method I
<1:20 2,3-cis >95% 40 18 3,4-trans Method II <1:20 2,3-cis
>95% 40 18 3,4-trans .sup.1 The method that prepared HH-F3a (by
Sephadex LH-20 chromatography) .sup.2 by dialysis
[0081] Since no polymeric compound from GP has been reported, the
polyphenolic compounds from another precious crassulaceous herb
Rhodiola rosea (golden root) are utilized as reference compounds
for structural identification of HH-F3a. R. rosea has been reported
to have polymeric proanthocyanidin (PAC). The structure of the main
compounds in the HH-F3a fraction was very similar to the
proanthocyanidin compounds of R. rosea (see Table 2) but with
rather minor signals (<5%) for a procyanidin unit (PC unit),
which was not detectable in the .sup.13C NMR spectrum
(.delta..sub.C 114 ppm, B ring C-2' and C-5'). In addition, the PAC
compounds are frequently found in many common grape species, such
as Vitis vinifera. Thus, the PACs from V. vinifera are also brought
into comparison. Table 2 shows the physiochemical properties for
the proanthocyanidin polymers from R. rosea and V. vinifera (a
common grape species). Compared to the data in Table 2, the
proanthocyanidin compound in the HH-F3a fraction was within
2.5.times. of the PD to PC ratio, 3.0.times. of the mDP and mMW for
R. rosea, and 30 to 80.times., 1.1 to 4.9.times., and 4.7 to
41.3.times. higher than the mDP, mMW and % of 3-O-galloyl for V.
vinifera. To the best of our knowledge, no proanthocyanidin
compound from GP has been isolated, and no proanthocyanidin
compound with identical physiochemical properties has been
reported. This evidence suggests that the proanthocyanidin compound
found in the HH-F3a fraction is a new compound that is rich in
3,4,5-trihydroxy benzylic moieties (including a B ring of the PD
unit and gallic acid), very similar to that found in R. rosea but
much higher than that found in grape skin and seed.
TABLE-US-00002 TABLE 2 Physicochemical Properties for Known
Proanthocyanidin Polymers 3-O-galloyl mMW Source PC:PD cis:trans
(%) mDP (kD) Rhodiola rosea 1:8 2,3-cis >95 13.3 6.0 3,4-trans
Seed of Vitis 4:1 2,3-cis 20.4 8.1 2.6 vinifera.sup.1 Skin of Vitis
3:2 2,3-cis 2.3 34.9 10.4 vinifera .sup.1The European grapevine
native to the Mediterranean and Central Asia
Example 3
Effect Examples
1. Viability Assay
[0082] The cells were seeded in 24-well plates (4,000-5,000
cells/well), incubated overnight and then treated with the 30% DMSO
GP extract or the HH-F3 fraction for 0, 24, 48, or 72 hours. After
treatment, the cells were gently washed 3 times with 1.times.PBS
(137 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO.sub.4, 2 mM
KH.sub.2PO.sub.4) and then were incubated with 0.5 .mu.g/mL
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
for 2 hours. The medium was removed, and the deep-blue crystals
were dissolved with 100% DMSO at room temperature for 10 minutes.
OD values were measured at 570 nm with an ELISA reader.
2. Western Blot
[0083] All samples were denatured by heating at 95.degree. C. for
10 minutes and resolved by 8% or 10% SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) at 80 V and 100 V for the stacking and
running gel, respectively. After SDS-PAGE, the proteins were
transferred to polyvinylidene difluoride (PVDF) membranes using the
Bio-Rad transfer system. After the proteins were transferred, the
membranes were stained with Ponceau S to confirm the efficiency and
uniformity of the protein transfer. The membranes were blocked with
5% non-fat skim milk (BD) at room temperature for 30 minutes and
then were incubated with primary antibody at 4.degree. C.
overnight. Afterward, the membranes were washed with
1.times.Tris-buffered saline Tween-20 (TBST) three times (10
minutes each). The membranes were incubated with secondary antibody
for 2 hours. Then, they were washed with 1.times.TBST three times
(10 minutes each). The signals of the secondary antibodies were
visualized by adding HRP substrate peroxide solution/luminol
reagents (Immobilon.TM. Western Chemiluminescent Substrate,
Millipore; mixed at a 1:1 ratio) and were detected by the Fujifilm
LAS4000 luminescent image analysis system.
3. Cell Counting
[0084] The cells were seeded in 12-well plates (10,000-30,000
cells/well) overnight and then were treated with the HH-F3 fraction
for 0, 24, 48, or 72 hours. The cells were trypsinized and then
counted after mixing with 0.4% trypan blue.
4. Cell Cycle Analysis and Flow Cytometry
[0085] After trypsinizing the cells and washing with 1.times.PBS 3
times, the cells were centrifuged at 800 g for 5 minutes. Then, the
cells were resuspended in 70% ethanol in PBS and kept at
-20.degree. C. for more than 16 hours. After centrifugation at 800
g for 5 minutes, the cell pellets were resuspended with cold PBS
containing 100 .mu.g/mL RNAse A (Sigma-Aldrich) for 20 minutes.
Then, the cells were stained with 20 .mu.g/mL propidium iodide (PI,
Sigma-Aldrich) for 20-30 minutes, and the DNA content was measured
by the BD FACSCanto and analyzed by FlowJo software.
5. Mitochondrial Membrane Potential Assay
[0086] Mitochondrial membrane potential was analyzed using
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine
iodide (JC-1), which was purchased from Cayman Chemical Co. The
cultured cells were seeded in 96-well black plates at a density of
7000 cells/well and incubated overnight, then treated with or
without the HH-F3 fraction for 48 hours. The JC-1 staining solution
was added to each well and mixed gently at 37.degree. C. for 15-30
minutes in the dark. The plates were centrifuged at 400 g at room
temperature for 5 minutes, and the supernatant was removed. Then,
JC-1 assay buffer was added to each well, followed by
centrifugation at 400 g at room temperature for 5 minutes, after
which the supernatant was removed. Finally, JC-1 assay buffer was
added to each well for analysis using a fluorescent plate
reader.
6. Measurement of ROS Levels
[0087] Intracellular generation of superoxide radicals
(O.sub.2.sup.-) was assessed by hydroethidine fluorescence (AAT
Bioquest, Inc.). The cells were treated with or without the HH-F3
fraction for 48 hours. Hydroethidine (10 .mu.M) was added to each
well and was mixed gently for 30-60 minutes at 37.degree. C. in the
dark. Cellular fluorescence was monitored at wavelengths of 520 nm
(excitation) and 610 nm (emission).
[0088] Intracellular peroxide levels were determined with
dichlorofluorescein (DCFH) diacetate (Marker Gene Technologies,
Inc.). Following treatment with the HH-F3 fraction for 48 hours,
the medium was aspirated, and the cells were washed twice with PBS.
Then, the cells were incubated with DCFH at a final concentration
of 20 tM in serum-free media for 30-60 min at 37.degree. C. in the
dark. The cells were washed again with PBS and maintained in 200
.mu.L of culture media. Cellular fluorescence was monitored at
wavelengths of 485 nm (excitation) and 528 nm (emission).
7. Animals and the Experimental Environment
[0089] A total of 120 male Wistar albino rats (150-180 g) that 6
weeks of age at the start of the experimental period were used. All
animals were fed ad libitum with standard chow and water during the
study and were acclimated for 7 days before disease induction.
Experimental Protocol
[0090] The rats were randomly divided into the normal group (N=10),
the diethylnitrosamine (DEN) group (N=30), the low-dose GP group
(N=30) and the high-dose GP group (N=30). We included another 5
rats for the HH-F3-treated group. In all groups except the normal
group, the rats drank an aqueous solution of 100 ppm (v/v) DEN
(Sigma-Aldrich, St. Louis, Mo., USA) daily as the sole source of
drinking water for 63 days, and starting on day 64, they were fed
tap water for another 14 days. DEN solution was prepared each week
and consisted of an individualized dose according to the weight
gain/loss of the animal in response to the previous dose. Visible
liver tumors were noted after day 42, and liver fibrosis was
observed after day 63. During the experimental period, the animals
were weighed weekly to calculate weight gain, and the amount of
water consumption was also measured every week. In the low-dose
group, the rats received 0.6 g/rat lyophilized GP powder, and in
the high-dose group, the rats received 1.8 g/rat lyophilized GP
powder daily beginning on day 42 for 3 weeks.
[0091] Harvesting Procedure and Morphologic Evaluation of the
Liver
[0092] All animals were euthanized on the day 84. The animals were
fasted overnight and then sacrificed by CO.sub.2 inhalation. After
the rats were sacrificed, the bodies, livers and spleens were
weighed, and the conditions of the organs were recorded after
necropsy, which followed a midline laparotomy. All lobes of the
liver were promptly harvested and thoroughly examined to clarify
the nature of the liver surface and the development of liver foci,
persistent nodules (PNs), or cancer at every time point;
subsequently, the liver was cut into 5-mm sections. All
macroscopically visible nodules were counted on the liver surface
and in the 5-mm sections to determine their number and size.
[0093] Tumor Burden Assessment
[0094] To establish the course of tumor development in the animals
fed with DEN, all lobes of the liver were promptly harvested, and
all macroscopically visible nodules were counted on the liver
surface and in the 5 mm sliced sections to determine their numbers
and sizes. Tumor burdens were determined by estimating the sums of
the volumes of all tumor nodules with diameters greater than 3 mm
for each animal and then comparing the groups.
[0095] Bile Flow Rate
[0096] Bile flow rate was measured prior to the sacrifice of the
animals after deep anesthesia with 80 mg/kg ketamine. To measure
the bile flow rate, a PE10 silicon tube was placed in the common
bile duct and then connected with a calculated polyethylene tube.
Bile flow in the tube was measured at 5-min intervals.
[0097] Histopathological Evaluation
[0098] After draining the blood, tissue slices of approximately
5-mm thickness that contained tumors were dissected from each lobe
of the liver. Sections with thicknesses of 5 .mu.m were cut and
stained with hematoxylin and eosin for histopathological analysis
using published diagnostic criteria.
[0099] Immunohistochemical Staining for .alpha.-Smooth Muscle Actin
(.alpha.-SMA)
[0100] The liver samples were fixed with formalin, embedded with
paraffin, and then sectioned into 5 .mu.m sections. The sections
were deparaffinized, rehydrated and then treated with 0.03%
hydrogen peroxide for 10 minutes to quench endogenous peroxidase
activity. Following two washes with PBS, the sections were
incubated for 1 h at room temperature with a mouse anti-human
.alpha.-SMA monoclonal antibody (1:50 dilution, DakoCytomation,
Denmark). For .alpha.-SMA staining, the sections were washed and
further incubated with a secondary antibody, rabbit anti-mouse IgG
(1:200 dilution), at room temperature for 1 hour. The sections were
then developed similarly. After staining, the sections were
counterstained with hematoxylin for microscopic examination. The
percentage of .alpha.-SMA-positive area (mm.sup.2/cm.sup.2 of liver
section) was measured using the Digital Camera System HC-2500 (Fuji
Photo Film), Adobe Photoshop version 5.0J, and Image-Pro Plus
version 3.0.1J.
[0101] Assay of Hydroxyproline Content in the Liver
[0102] Liver specimens were weighed, and 20 mg of the frozen
samples was hydrolyzed in 20 mL of 6 N HCl and carefully ground.
Additionally, 6 N HCl was then added to obtain a total volume of 30
mL per mg tissue. The ground tissue in HCl was hydrolyzed at
120.degree. C. for 16 h. After brief cooling on ice and
centrifugation at 8000 g for 10 min, the supernatant was removed
and placed in a new tube; the volume lost to evaporation was
replenished by water. An equal volume of 6 N NaOH was added and
mixed, and the solution was adjusted to pH 4-9 using litmus paper.
Forty microliters of the neutralized sample solution was added to
the wells of a 96-well ELISA plate and oxidized using a solution
containing 5 mL of 7% chloramine T (Sigma-Aldrich) and 20 mL of
acetate/citrate buffer. Thereafter, 150 mL of Ehrlich's solution
was added. The final mixture was incubated at 60.degree. C. for 35
min and then at room temperature for another 10 min, after which
the absorbance was determined at 560 nm. Standard solutions
containing 100, 80, 60, 40, 20 and 0 mg/mL of authentic
4-hydroxy-L-proline (Sigma-Aldrich) were treated likewise. The
standard curve was linear in this range (r=0.99). The value of the
liver hydroxyproline level was expressed as hydroxyproline (mg)/wet
liver weight (g). All assays were repeated in triplicate.
[0103] Immunocytochemistry for Oxidative Stress
[0104] A nitroblue tetrazolium (NBT, Sigma-Aldrich) perfusion
method was used for localizing de novo ROS generation in the liver.
NBT-perfused livers were removed and fixed in a zinc/formalin
solution and processed for histological examination of formazan
deposits. The density of blue NBT deposits was determined using
Adobe Photoshop 7.0.1 image software analysis.
[0105] Results
[0106] The Effects of Different GP Preparations on Huh7 and Mahlavu
Cells
[0107] To test the potential biological effects of GP, different
preparations of GP extracts, including water extract, butanol
extract, acetone extract, methanol extract, 100% ethanol extract,
70% ethanol extract, 50% ethanol extract, 100% DMSO extract and 30%
DMSO extract were prepared and used to treat human HCC cells. As
shown in FIG. 1, the growth inhibitory effects that were caused by
different preparations of the GP extracts were evaluated in a
dose-dependent manner. The MTT assay results indicated that the 30%
DMSO extracts significantly inhibited the viability of Huh7 (FIG.
2) and Mahlavu (FIG. 2) cells.
[0108] The GP reduced AURKA, AURKB and FLJ10540 protein expression
levels during both interphase and mitosis in activated hepatic
stellate cells and HCC cell lines.
[0109] AURKA, AURKB and FLJ10540 are oncogenes and are
overexpressed in HCC. Therefore, we tested whether the different
preparations of GP extracts inhibit these oncoproteins in human HCC
cell lines. We found that the GP extracts (obtained from 100%
methanol followed by 30% DMSO extraction, as described in the
methods section and referred to as 30% DMSO GP extracts) inhibited
the protein expression levels of FLJ10540 and AURKB in activated
hepatic stellate cells (HSC-T6) (FIG. 3) and suppressed the protein
expression levels of FLJ10540, AURKA, and AURKB in HCC cells (HepG2
and Huh7) (FIG. 3). The expression levels of both AURKA and
FLJ10540 are higher during metaphase than interphase. We next
investigated whether GP inhibits the expression of these two
proteins during mitosis in HCC cells. Huh7 and HepG2 cells were
treated with 50-75 ng/mL nocodazole for 18 hours, followed by
treatment with the 30% DMSO GP extracts for 3 hours without washing
out the nocodazole. Consistent with previous findings, AURKA,
AURKB, and FLJ10540 were highly expressed during mitosis. The
protein expression levels of AURKA, AURKB, and FLJ10540 were
decreased during both interphase and metaphase (FIG. 3), whereas no
significant changes were observed in the other mitotic proteins
that we examined, including PINT, HURP, and PLK (data not
shown).
[0110] The 30% DMSO GP extracts (the fraction HH-F3), but not the
other extracts that were prepared from different solvent,
suppressed AURKA protein expression in HCC cell lines.
[0111] Human HCC Huh7 cells were treated with 500 .mu.g/mL of the
different preparations of GP extracts for 48 hours. The 30% DMSO
extract significantly inhibited the protein expression levels of
AURKA in these cells (FIG. 4). In contrast, the GP extracts
obtained from either water or butanol did not inhibit the protein
expression levels of AURKA or AURKB in HepG2 cells after 3 hours of
treatment (FIG. 4). Because AURKA and FLJ10540 are overexpressed in
HCC, we examined whether GP has an effect on the growth of HCC
cells. We found that the 30% DMSO GP extracts caused cytotoxicity
of Huh7 and Mahlavu cells with a 50% inhibitory concentration of
cell viability (IC.sub.50), which was determined to be
approximately 500 and 250 .mu.g/mL at 48 hours post-treatment,
respectively (FIG. 5).
[0112] HH-F3 Suppressed AURKA Protein Expression in HCC Cell
Lines
[0113] The 30% DMSO GP extracts reduced the protein expression
levels of AURKA and F1110540 in activated hepatic stellate cells
and hepatoma cells (FIG. 2), suggesting that we could use this
assay to purify the active molecule(s) in GP. Using a Sephadex
LH-20 column, we obtained four fractions from the 30% DMSO GP
extracts (FIG. 6). Only the third fraction (referred to as HH-F3)
suppressed the expression of AURKA and AURKB in HepG2 cells, as
examined by Western blot at 3 hours post-treatment, while the other
fractions (HH-F1, HH-F2 and HH-F4) did not inhibit AURKA and AURKB
protein expression (FIG. 6). Taken together, GP extracts prepared
from 30% DMSO and the HH-F3 fraction inhibited the protein
expression levels of AURKA and AURKB in HepG2 cells at 3 hours
post-treatment (FIG. 6). We further obtained an active subfraction
from HH-F3, which is referred to as HH-F3a (with a yield of 71.9%
compared to the amount of the starting material HH-F3), using
dialysis. This HH-F3a fraction, but not HH-F3b fraction, contained
the active compounds, as determined by measuring the disappearance
of AURKA by Western blot (FIG. 6).
[0114] The HH-F3 Fraction Reduces Cell Viability in HSC-T6 Cells
and HCC Cell Lines
[0115] To investigate the effects of the HH-F3 fraction on cell
viability, Huh7, Mahlavu, PLC5, and HSC-T6 cells were treated with
the HH-F3 fraction at concentrations of 5, 25, 50, 75, and 100
.mu.g/mL for 24, 48, and 72 hours. The survival of these tested
cell types were inhibited in response to HH-F3 treatment, as
examined by the MTT assay. The IC.sub.50 values for treatment with
the HH-F3 fraction in Huh7, Mahlavu, PLC5, and HSC-T6 cells at 72
hours were approximately 50, 37.5, 75, and 20 .mu.g/mL,
respectively (FIG. 7). To further confirm this finding, cell
survival was determined by trypan blue staining of Huh7, Mahlavu,
and PLC5 cells. Decreases in cell survival after treatment with the
HH-F3 fraction were demonstrated in a time- and dose-dependent
manner after 24, 48, and 72 hours at concentrations of 5, 25, 50,
75, and 100 .mu.g/mL (FIG. 7). Taken together, both the 30% DMSO GP
extracts and the HH-F3 fraction can inhibit the cell viability of
HCC cell lines and activated hepatic stellate cells.
[0116] Next, Huh7, Mahlavu, PLC5, and HSC-T6 cells were treated
with 25, 50, and 75 .mu.g/mL of the HH-F3 fraction for 3 hours. The
HH-F3 fraction suppressed the expression of both AURKA and FLJ10540
in all three tested HCC cell lines and HSC-T6 cells (FIG. 8). To
investigate whether the inhibitory effects of the HH-F3 fraction
occurred at the transcriptional level, we examined the variation in
the gene expression levels of FLJ10540 and the Aurora kinase family
(AURKA, AURKB, and AURKC). HepG2 cells were treated with 50
.mu.g/mL of the HH-F3 fraction for 6 hours, after which gene
expression levels were analyzed by microarray (U133A chip,
Affymetrix), and protein levels were analyzed by Western blot.
Compared with the control group, there was no change in the gene
expression levels of the specific genes mentioned above after
treatment with the HH-F3 fraction (data not shown), despite a
decrease in protein levels. Therefore, the HH-F3 fraction probably
regulates HCC cell growth at the protein level and not at the
transcriptional level.
[0117] The HH-F3 Fraction Leads to Cell Death Via Apoptosis in HCC
Cell Lines
[0118] The extracts were analyzed for the effects of the HH-F3
fraction on the cell cycle profiles of HCC cells using propidium
iodide (PI) staining Huh7 and Mahlavu cells were treated with 5,
25, and 50 .mu.g/mL HH-F3 for 48 hours. The HH-F3 fraction
disturbed the cell cycle progression of Huh7 and Mahlavu cells.
After 48 hours of treatment with 50 .mu.g/mL of the HH-F3 fraction,
the sub-G1 population of Huh7 was 22%, while it was 26% in Mahlavu
cells. The HH-F3 fraction generated a larger increase in the sub-G1
population in Mahlavu cells than in Huh7 cells, which is in
accordance with the cytotoxic effects demonstrated earlier (data
not shown). We next examined the ability of the HH-F3 fraction to
induce apoptotic cell death in Huh7 and Mahlavu cells. The protein
expression levels of cleaved caspase-3 and cleaved PARP were
increased in a dose-dependent manner at concentrations of 5, 25,
and 50 .mu.g/mL for 24 and 48 hours. Under the same concentration,
HH-F3 fraction also resulted in the up-regulation of apoptotic
molecule FAS and the down-regulation of BCL2 and BCL-XL (data not
shown). These data indicate that the HH-F3 fraction induces
caspase-dependent apoptotic cell death (FIG. 9).
[0119] The HH-F3 Fraction Decreases Mitochondrial Membrane
Potential and Increases ROS in HCC Cell Lines
[0120] Reactive oxygen species (ROS) and mitochondria play an
important role in apoptosis induction under both physiological and
pathological conditions. We next investigated whether the HH-F3
fraction triggers apoptosis via the extrinsic or intrinsic pathway.
We tested whether mitochondrial membrane potential, one of the
indicators of the intrinsic pathway, might be altered in HCC cells.
Huh7 and Mahlavu cells were treated with 5, 25, and 50 .mu.g/mL of
the HH-F3 fraction, and the mitochondrial membrane potential of the
cells was examined after 48 hours of treatment. Compared to the
control group, the number of apoptotic cells was increased. This is
in agreement with mitochondrial membrane potential (AT) results,
which show that the membrane potentials were decreased in Huh7 and
Mahlavu cells after treatment with the HH-F3 fraction (FIG.
10).
[0121] Several reports have shown that ROS are generated only after
the loss of .DELTA..PSI.. ROS include superoxide anions, hydrogen
peroxide, and hydroxyl radicals, all of which are derived from
oxygen. ROS are produced as a consequence of electron transport
processes during photosynthesis and aerobic respiration. ROS, at
the physiological concentrations required for normal cellular
function, are involved in intracellular signaling and redox
regulation. Excessive levels of ROS cause oxidative stress, which
is potentially harmful to cells because it causes the oxidation of
lipids, proteins and DNA. We tested whether stimulation of the HCC
cells with the HH-F3 fraction would result in changes in the
production of ROS. Intracellular generation of O.sub.2.sup.- was
assessed by hydroethidine fluorescence, and the level of
intracellular peroxide was determined with DCFH diacetate. After
the cells were treated with the HH-F3 fraction, cellular production
of intracellular peroxide (FIG. 10) and superoxide (FIG. 10) were
increased in HCC cells. This suggests that the HH-F3 fraction
causes apoptosis via the intrinsic pathway.
[0122] The HH-F3 Fraction Decreased the Phosphorylation of Akt and
Enhances the Expression of PTEN
[0123] Some cell proliferation pathways are related to apoptosis
inhibition and abnormality in HCC, for example AKT pathway. Because
HH-F3 caused cell cytotoxicity on HCC cells, we then investigated
whether HH-F3 affected the cell proliferation pathways on HCC
cells. The Huh7 cells were treated with HH-F3 at 5, 25, 50 .mu.g/mL
for 48 hours, respectively. In Huh7 cells, the Ser.sup.473
phosphorylation of AKT was down-regulated under HH-F3 treatment,
whereas the total AKT protein was not influenced (FIG. 11).
Interestingly, HH-F3 activated the protein level of phosphatase and
tensin homolog (PTEN), which is a negative regulator of PI3K/AKT
dependent signaling. These results indicated that HH-F3 may
modulate AKT signaling transduction pathway of cell proliferation
to induce cell apoptosis.
[0124] GP Extracts Increased Bile Excretion Function of Cirrhotic
Animals
[0125] Liver cirrhosis was also evaluated by measuring bile flow
rates, reflecting liver function (FIG. 12), by quantifying the
ratios of spleen weight/body weight, an indicator due to
cirrhosis-related portal hypertension (FIG. 12), and by analyzing
the expression of .alpha.-SMA induced by DEN (FIG. 12). All of the
data demonstrated that the status of liver cirrhosis was improved
after treated by high-dose GP, and presented as increasing bile
flow, decreased spleen size and decreased the percentages of
.alpha.-SMA (+) area significantly.
[0126] GP Extracts and HH-F3 Decrease the Hydroxyproline Content in
Cirrhotic Liver
[0127] Liver fibrosis was determined by measuring the levels of
liver hydroxyproline content. Significant increases of
hydroxyproline level were observed in DEN-induced animals
(143.+-.30 4/g). In contrast, following treatment with low dose GP,
high dose GP or HH-F3, the hydroxyproline contents were 98.+-.18
.mu.g/g (P<0.05 compared with the DEN group), 70.+-.10 .mu.g/g
(P<0.05), and 72.+-.8.2 .mu.g/g (P<0.05), respectively (FIG.
12).
[0128] GP Extracts and HH-F3 Decreased Oxidative Stress
[0129] NBT (Nitrotetrazolium blue chloride) is a dye that is
reduced to an insoluble blue-colored formazan derivative upon
exposure to superoxide, and the blue-colored deposit as a
histological marker for the presence of superoxide in tissue is
detectable by light microscopy. The density of NBT (+) foci was
determined from the 10 fields with the densest staining Significant
increases of NBT (+) foci were observed in DEN-induced animals
(23.+-.3). In contrast, following treatment with low dose GP, high
dose GP or HH-F3, the density of NBT (+)foci were 13.+-.4
(P<0.05 compared with the DEN group), 4.2.+-.0.6 (P<0.005),
and 6.2.+-.2.1 (P<0.05), respectively (FIG. 12). These findings
suggest that the oxidative stress induced by DEN could be reduced
by the treatment of GP extracts and HH-F3.
[0130] GP Extracts and HH-F3 Decreased the Tumor Burdens
[0131] The livers obtained from the sacrificed animals were sliced
into 5-mm sections. The numbers and sizes of all visible tumor
nodules with diameters larger than 3 mm were counted and measured.
Tumor burdens are expressed as the sum of the volume of total tumor
nodules. Visible tumors were observed in DEN-induced animals (tumor
burden 2350.+-.905 mm.sup.3. In contrast, following treatment with
low dose GP, high dose GP or HH-F3, the tumor burden in liver were
110.+-.105 mm.sup.3 (P<0.005 compared with the DEN group),
23.+-.31 mm.sup.3 (P<0.005), and 86.+-.12 (P<0.05),
respectively (FIG. 12). Representative photographs of the livers
showed multiple hepatic tumors in the cirrhotic rat livers. The
development of granulation on the surface and the uneven boundary
with multiple hepatic tumors was observed in these animals, and the
visible tumor number and uneven liver surface were improved after
treatment of low dose GP, high dose GP or HH-F3.
[0132] The Rhodiola rosea extracts were also tested and it was
found that they inhibited cell viability of the HCC cell lines and
down-regulate AURKA protein expression (FIGS. 13 (cell viability)
and 13 (down-regulation of AURKA protein expression)).
[0133] It is believed that a person of ordinary knowledge in the
art where the present invention belongs can utilize the present
invention to its broadest scope based on the descriptions herein
with no need of further illustration. Therefore, the descriptions
and claims as provided should be understood as of demonstrative
purpose instead of limitative in any way to the scope of the
present invention.
* * * * *